Human Tumor–infiltrating Lymphocytes Transfected with Tumor Necrosis Factor Gene Could Augment Cytotoxicity to Autologous Tumor Cells

Human tumor–infiltrating lymphocytes (TILs) derived from pleural or ascitic fluid were incubated with recombinant interleukin 2 and transfected with human tumor necrosis factor (TNF) a gene by the lipofection procedure. The resulting TILs secreted significant amounts of TNF in the culture supernatant and exhibited cytotoxicity against established cell lines, such as K562 and Daudi, and autologous tumor cells. The TNF gene–transfected TILs exhibited an augmented killing of autologous tumor cells.     

Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method.

Using differential radiation sensitivity of components of mouse embryonal thymus, an in vitro method for studying T-cell differentiation from hemopoietic stem cells (HSCs) in the G0 phase was established. Intrathymic T-cell precursors present in embryonal thymus were found to be quite radioresistant (up to 20 Gy), and consequently 25-Gy-irradiated embryonal thymic lobes were used. Thymic lobes (25-Gy irradiated) taken from mouse fetuses (gestation day 15) were placed in Millipore-HA culture plates supported on squares of gelatin foam sponge in 24-well culture plates in which neonatal thymus stromal cells were cultured. HSCs (10(5) cells per well) in the G0 phase were added to these thymic lobes and cocultured at 37 degrees C in a 5% CO2/95% air incubator. Half the culture medium was changed every week. After 3 weeks, a large number of colonies had formed. Immunohistochemical studies and fluorescence-activated cell sorter analyses revealed that the colonizing cells regularly develop and exhibit surface markers characteristic of T cells (Thy-1, IL-2R, L3T4, Lyt-2, etc.). In situ hybridization analyses revealed that mRNA expression for T-cell receptor (TCR) beta chains occurred within colonizing cells. Using a monoclonal antibody (F23.1), expression of TCR beta-chain variable domain (V beta 8) on the surface of these developing T cells was demonstrated. These cells responded to interleukin 2 and/or anti-CD3 monoclonal antibody, indicating functional T cells. This method will be useful in studying T-cell differentiation pathways from pluripotent HSCs and in clarifying the mechanisms involved in negative and positive selection of T cells within the thymus.     

Mercury-selenium interaction in workers exposed to elemental mercury vapor

Mercury (Hg) and selenium (Se) levels were determined for erythrocytes, plasma and urine of 25 male and 29 female workers exposed to elemental mercury (Hgo) vapor. Interrelationship between the Hg levels and the Se levels was examined by the correlational analysis and the stepwise regression analysis. Indicators of Se status (erythrocyte Se levels, plasma Se levels and urinary Se levels) were closely intercorrelated; similarly, indicators of exposure to Hgo vapor (erythrocyte inorganic Hg levels, plasma Hg levels and urinary Hg levels) were significantly intercorrelated. Since plasma Hg level was one of the significant independent variables in determining the erythrocyte Se and plasma Se levels, Hgo vapor exposure was thought likely to influence the Se metabolism in workers. None of the Se status indicators were significant as determinants of the indicators of Hgo vapor exposure.     

Split tolerance between spleen and lymph node cells in severe combined immunodeficiency mice grafted with AKR fetal liver cells

Severe combined Immunodeficient (SCID) mice defective in stemcells for T and B cells appear to be an ideal host for constructionof chimeric mice. When bone marrow cells are used as a sourceof stem cells, however, host SCID mice do not always show sufficientreconstitutlon. In this study, fetal liver cells from AKR embryoswere transplanted into SCID mice without prior irradiation.This treatment induced full reconstltution of lymphopoiesisas evaluated by flow cytometry analysis and serum Ig production2 months after transplantation. Thus, fetal liver cells seemto be a better source for reconstitutlon of SCID mice than bonemarrow cells. Lymph node (LN) cells of these mice (FLT mice)had no proliferatlve or cytotoxlc activities against eitherhost-type (C.B-17) or donor-type (AKR) spleen cells. However,spleen cells from FLT mice exhibited marked proliferatlve andcytotoxlc activities against C.B-17 cells, with no activitiesagainst AKR cells. Spirt tolerance against C.B-17 cells In spleenand LN cells was not a transient phenomenon, since similar resultswere obtained from a cytotoxic T lymphocyte assay 4 months later.In spite of the strong host reactivity in vitro, aberrationof clonal deletion or development of a graft-versus-host diseasewas not seen in FLT mice. As IL-2 induced the host reactivityof LN cells in a mixed lymphocyte reaction, potentially host-reactiveT cells were present in LN but were rendered anerglc. Tolerancein FLT mice seems to be regulated by a peripheral mechanism.We supposed that the split tolerance in FLT mice was inducedby the different antigenicity between the spleen and LN.     

WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors and is essential for resistance to Leishmania major infection. In the present study, we demonstrated that WSX-1 was also required for resistance to Trypanosoma cruzi. WSX-1-/- mice exhibited prolonged parasitemia, severe liver injury, and increased mortality over wild-type mice. WSX-1-/- splenocytes produced enhanced levels of Th2 cytokines, which were responsible for the prolonged parasitemia. Massive necroinflammatory lesions were observed in the liver of infected WSX-1-/- mice, and IFN-gamma that was overproduced in WSX-1-/- mice compared with wild-type mice was responsible for the lesions. In addition, vast amounts of various proinflammatory cytokines, including IL-6 and TNF-alpha, were produced by liver mononuclear cells in WSX-1-/- mice. Thus, during T. cruzi infection, WSX-1 suppresses liver injury by regulating production of proinflammatory cytokines, while controlling parasitemia by suppression of Th2 responses, demonstrating its novel role as an inhibitory regulator of cytokine production.     

Expression of lysosome-associated membrane proteins in human colorectal neoplasms and inflammatory diseases

The lysosome-associated membrane proteins (LAMPs)-1 and -2 are major constituents of the lysosomal membrane. These molecules are known to be among the most glycosylated proteins of several types of cells and cancer cells, and their expression in cancer cells is marked by a distinct difference in the structures of the oligosaccharides as compared to nonmalignant cells. We analyzed by immunohistochemistry the intensity and distribution of LAMP-1 and LAMP-2 in 9 human colorectal cancer cases and in 16 control cases, including inflammatory diseases (diverticulitis, ulcerative colitis, and Crohn's disease). LAMP proteins were expressed more intensely in the epithelium of colorectal neoplasms than in normal mucosa (P < 0.05), and no significant differences were found between adenoma and cancer cells (P > 0.05) in the same tissue section. Further, in sites of inactive inflammatory diseases and nonneoplastic areas in cancer specimens, no significant increases in epithelial LAMP proteins were observed, even in the proliferative zone of the lower crypt epithelium. Northern blot analysis showed increased expression of LAMP-1 and LAMP-2A in two of three colorectal cancers examined and increased LAMP-2B in all three cancers. Our findings suggest that LAMPs are related to neoplastic progression, but there is no direct association between the expression of LAMP molecules and cell proliferation.     

Bone marrow-derived progenitor T cells convey the origin of maturational arrest from CD4+CD8+ to CD4-CD8+ thymocytes in LEC mutant rats.

A mutant strain of rats, LEC, shows a novel arrest of T cell maturation from CD4+CD8+ to CD4+CD8- but not to CD4-CD8+ cells in the thymus. Transplantation of LEC rat fetal thymuses into the subcapsule of the kidney of athymic nude rats resulted in a normal maturation of thymocytes in the thymus graft. Furthermore, both single-positive thymocytes and peripheral lymph node T cells expressed T cell receptor alpha/beta antigen, and lymph node T cells acquired the ability to produce interleukin 2 upon mitogen stimulation. Transplantation of fetal thymuses from LEA rats, which express the same major histocompatibility complex haplotype as LEC rats, into LEC rat kidney subcapsule resulted in the maturational arrest from CD4+CD8+ to CD4+CD8- cells in the thymus graft. These data strongly suggest that bone marrow-derived progenitor T cells carry the cause of maturational arrest and that the thymic stroma of LEC rats has a normal potential to nurse thymocytes.     

Comparability of delayed hypersensitivity in various rodents. II. Jones-Mote type hypersensitivity in guinea-pigs immunized with sheep erythrocytes and its modification by cyclophosphamide or BCG pre-treatment.

Guinea-pigs were immunized via footpads with sheep red blood cells (SRBC) in saline. Histological examination of erythematous skin reaction was performed and effects of cyclophosphamide (CY) or BCG pre-treatment on the skin reaction were examined. Delayed-in-onset erythematous skin reaction accompanied by substantial basophil infiltration was elicited in guinea-pigs immunized with SRBC in saline. The erythema was augmented in size by CY which was injected 2 days before immunization. The reaction may be comparable to Jones-Mote type. In BCG pre-treated guinea-pigs, basophil infiltration at the skin reaction sites was reduced in number, but significant inhibition of macrophage migration was not detected in the presence of SRBC antigen. The reaction may be intermediate between Jones-Mote and the tuberculin type. Comparability of delayed skin reactions in guinea-pigs and delayed footpad reactions in mice or hamsters against SRBC is discussed.     

Specific atrial natriuretic peptide binding sites in rat cerebral capillaries

We examined the specific rat 125I-alpha-rat atrial natriuretic peptide(1-28)[ANF-(99-126)] (125I-rANP) binding sites in the cerebral capillaries from the cerebral cortex of male adult Wistar rats. The binding of 125I-rANP at 37 degrees C was saturable and of high affinity with a Kd of approximately 100 pM and Bmax of 152 fmol/mg protein. Divalent cations, Mn2+ (2.2 mM) and Ca2+ (1.8 mM) potently inhibited the binding. The rank order for inhibition of the binding was rANP, alpha-human ANP and ANF-(101-126) greater than ANF-(103-126) and ANF-(103-125)"ANF-(103-123). These data on specific binding sites of ANP in cerebral capillaries suggest a possible role for ANP in the blood-brain permeability of water and electrolytes.     

Practical application of nutritional assessment protein and inflammatory marker in the nutrition support team (NST)

Recently nutrition support team (NST) has been established for the purpose of prevention of complications which are caused by nutrition disorders and reduction of the medical expenses. Although physical examinations and blood biochemical data had been used as the indexes evaluating nutritional of patients, they were not suitable for the evaluation for the short-term in-patient. On the contrary, serum albumin (ALB) has been wildly used as a nutritional marker. However, it is impossible to evaluate nutrition state for the short-term in-patient and acute phase disease patient accurately, because the plasma half-life is 21 days and it takes long time to detect the change in nutritional state by its value. Rapid turnover proteins (RTP), whose plasma half-life is shorter, has paid attention to evaluate nutritional state for the short-term in-patients and acute phase disease patients. Although, prognostic inflammatory and nutritional index (PINI) was considered as a useful maker for evaluating inflammatory and nutritional states using the concentrations of transthyretin (TTR), a RTP, alpha1-acid glycoprotein (alpha1-AG), a chronic inflammation marker, C reactive protein (CRP), a acute inflammation marker, and ALB, However, it has several pitfalls. We newly made serum amyloid A (SAA) index using SAA instead of CRP. When we compared SAA index with PINI in many diseases, it turned out that SAA index became a more effective index which reflected the patient condition than did PINI. As for this index, it is expected to be used by NST while further alternation may be needed.